The long term goal of this project is to elucidate the role of aldehyde dehydrogenases (ALDH3A1 and ALDH1A1) in the ocular defense mechanism against ultraviolet radiation (UVR) toxicity. It is well known that UVR induces reactive oxygen species (ROS) that leads to membrane lipid peroxidation and accumulation of toxic aldehydes, such as 4-hydroxy-2- nonenal (4-HNE). Both ALDH3A1 and ALDH1A1 detoxify 4-HNE, which has been implicated in the pathogenesis of several diseases including cataract and degenerative retinal disease. During the last funding period, we have made novel and significant discoveries regarding the protective role of ALDH3A1 and ALDH1A1 against ocular oxidative stress. We found that both enzymes protect against UVR- and 4-HNE-induced cellular toxicity, and that, at least ALDH3A1, may act as UVR filter protecting other enzymes from inactivation. Finally, and most importantly, we found that Aldh3a1(-/-) and Aldh1a1(-/- )/Aldh3a1(-/-) knockout mice develop anterior and posterior subcapsular cataracts early in life, whereas Aldh1a1(-/-) knockout animals develop cataracts later in life. These novel data support our working hypothesis that corneal ALDH3A1 and lens ALDH1A1 protect the eye against cataract formation via non-enzymatic (light filtering) and enzymatic (detoxification) functions. The experiments proposed in this competitive renewal application continue our systematic approach to characterizing the role of corneal ALDH3A1 and lens ALDH1A1 in the protection against UVR- induced eye pathologies. In the next 5 years, we propose to: Specific Aim 1: To distinguish the physiological functions of non-enzymatic (light filtering) from enzymatic (detoxification) functions of ALDH3A1 and ALDH1A1 by developing Aldh3a1 and Aldh1a1 knock-in mouse lines that express enzymatically- inactive proteins. The ocular phenotypes of these knock-in mouse models will be compared to that of the Aldh3a1(-/-) and Aldh1a1(-/-) knockout mice and wild-type animals Specific Aim 2: Examine the hypothesis that novel and selective small molecule activators of ALDH1A1 and ALDH3A1 will protect the eye against UVR-induced pathologies. Following administration of these activators and exposure to UVR, corneal injury and cataract formation will be studied in parallel with formation and characterization of 4-HNE-adducted proteins in wild-type and Aldh-knockout mice. Specific Aim 3: Evaluate the hypothesis that ALDH3A1 and ALDH1A1 protect the retina. The protective effects of both ALDH3A1 and ALDH1A1 will be determined by identifying aging- and UVR-induced biochemical and histological changes in the retina of Aldh3a1(-/-), Aldh1a1(-/-) and Aldh3a1(-/-)/Aldh1a1(-/-) single and double knockout mouse lines. These studies will provide valuable data on (a) the role and the molecular mechanisms of the ocular ALDH1A1 and ALDH3A1 in protecting the eye from UVR-induced oxidative damage and (b) drug development that may be used to prevent eye pathologies. It is anticipated that the information gleaned from these studies will lead to novel approaches for the prevention and treatment of eye diseases in the human population.